The study of simian immunodeficiency virus (SIV) infection in macaques has provided numerous insights into the pathogenesis of AIDS in human immunodeficiency virus type 1 (HIV-1) -infected humans. However, gene structure and, consequently, some regulatory and structural features of the HIV-1 and HIV-2/SIV families are constitutively different. SIV/HIV-1 envelope chimeras (SHIV) mitigate some of the concerns about differences in immune responses and tropism directed by envelope proteins but the gag and regulatory genes in the SIV backbone compromise their usefulness for addressing mechanisms of pathogenesis and anti-viral strategies unique to HIV-1 gag and regulatory genes. Several laboratories have constructed chimeric SHIVS on the SIVmac backbone that are infectious for NHPs. In these constructs, SIV genes other than the envelope gene, i.e., LTR, gag, pol, vif, vpx, vpr, tat, rev and nef genes, appear to influence SHIV replicative potential in NHPs, but they are not yet well understood. Since the nef genes of HIV-1 and SIV play an important role in viral infectivity and pathogenicity, we generated HSIV nef chimerae. The nef gene of cloned HIV-1pNL4-3 was precisely exchanged with that of SIVmac239 to generate the nef gene, chimeric HSIV. The nef genes of HIV-1 and SIV overlap with their 3'LTR U3 regions and two different nef chimerae, NF-1 and NF-2, were constructed. The HSIV NF-1 chimera HIV-1 was constructed by replacing all the HIV-1 nef coding regions, including the 3'LTR U3 with the corresponding SIVmac239 regions. Therefore, NF-1 contains a full-length SIV nef coding region and a hybrid 3'LTR. To construct the NF-2 chimera, only the HIV-1 nef unique region not overlapping with the U3 of the 3'LTR (from nef amino acid #1 through #96) was exchanged with the nef unique region of SIVmac239. Therefore, the NF-2 chimera encodes a hybrid nef protein between HIV-1 and SIV (N-terminus encodes SIV nef and the C-terminus encodes HIV-1 nef) and retains the wild-type HIV-1 3'LTR. Both the NF-1 and NF-2 chimerics are fully infectious for human T cell lines, and their replicative potential in human T cell lines was indistinguishable from wild-type HIV-1pNL4-3. The replicative potential of the nef chimeric viruses in PBL from humans and several macaque PBMC will be further examined in culture. If the nef chimerics exhibit a significant replicative potential in macaque PBMC in culture, experimental macaque studies and serial passage will be initiated to evaluate in vivo infectivity and pathogenicity. FUNDING NIH RR-00163 (Project 7) PUBLICATIONS None